The discovery of a conserved pathway for endocytic internalization in budding yeast, a simple organism amenable to powerful genetic, genomic, biochemical and cell biological analysis, has created unprecedented opportunities to develop a comprehensive understanding of this complex process, and to reveal underlying molecular mechanisms. Endocytosis is a key process for specifying the molecular composition of the plasma membrane, and therefore how a cell will respond to its environment. Endocytosis also provides a portal for entry of both beneficial molecules and harmful agents into cells. Despite the importance of endocytosis for cell physiology and human health, many fundamental features of the process are obscure. How sites are initiated and what mechanisms trigger the subsequent events, which invaginate the membrane and pinch off a vesicle, are not known. In this proposal, an integrated approach, combining live-cell image analysis, genetics, mathematical modeling and biochemistry is outlined. These studies promise to elucidate molecular mechanisms that underlie the endocytic process. Two aims will be investigated. (1) How endocytic sites are initiated, matured and post-translationally regulated will be determined. The yeast Eps15-like protein, Ede1, which is among the earliest to arrive at endocytic sites, and which is among the most important proteins for endocytic site initiation and progression, will be a focal point. How Ede1 is recruited to nascent endocytic sites, and how it in turn recruits other proteins to these sites, will be investigated by a combined molecular-genetic, imaging and biochemistry approach. Roles for Ede1 phosphorylation by the novel endocytic kinase Hrr25 and for Ede1 ubiquitination, will be investigated. If, and how, these regulatory inputs are influenced by endocytic cargo will also be tested. Finally, how the rhomboid protease Rbd2 regulates the transition from the early to late stages of the endocytic pathway will be investigated. (2) The roles of specific plasma membrane lipids and protein-lipid interactions during each stage of the CME pathway will be elucidated. Endocytosis involves a series of membrane shape changes. A variety of endocytic proteins associate intimately with the lipid bilayer during each stage of the process, responding to and affecting lipid composition and membrane geometry. The specific roles and protein interactions of PS, PIP2 and sphingolipids will be investigated. Purified endocytic proteins and a recently developed cell extract system that reconstitutes the actin assembly burst near the end of the endocytic pathway will now be used to reconstitute endocytic steps on microbeads and lipid bilayers, establishing biochemical systems to reveal mechanism and to complement and synergize with powerful genetic and imaging approaches. A highly productive theory collaboration will continue to facilitate data synthesis and development of novel mechano-chemical concepts, and to generate experimentally testable hypotheses.